© 2019. Published by The Company of Biologists Ltd | Journal of Cell Science (2019) 132, jcs223123. doi:10.1242/jcs.223123

SHORT REPORT Chk1-mediated Cdc25A degradation as a critical mechanism for normal progression Hidemasa Goto1,*, Toyoaki Natsume2,3, Masato T. Kanemaki2,3, Aika Kaito4, Shujie Wang1, Esteban C. Gabazza5, Masaki Inagaki4 and Akira Mizoguchi1

ABSTRACT ATM is primarily activated by DNA double-strand breaks Chk1 (encoded by CHEK1 in mammals) is an evolutionarily conserved (DSBs). ATM phosphorylates and activates Chk2. Both TP53 that transduces checkpoint signals from ATR to Cdc25A phosphorylate (encoded by ) resulting in p53 stabilization, during the DNA damage response (DDR). In mammals, Chk1 also which promotes (also known as CDKN1A) expression. p21 controls cellular proliferation even in the absence of exogenous DNA binds and inhibits the and cyclin-dependent kinase (CDK) damage. However, little is known about how Chk1 regulates complexes leading to cell cycle arrest. By contrast, ATR is activated unperturbed cell cycle progression, and how this effect under by a broader spectrum of DNA-damaging stimuli, such as ultraviolet physiological conditions differs from its regulatory role in DDR. Here, (UV) radiation, DNA replication stress and inter-strand DNA we have established near-diploid HCT116 cell lines containing crosslinking reagents. ATR phosphorylates and activates Chk1, endogenous Chk1 protein tagged with a minimum auxin-inducible which in turn phosphorylates and inhibits . degron (mAID) through CRISPR/Cas9-based editing. Cdc25 dephosphorylates inhibitory phosphorylation sites on CDK Establishment of these cells enabled us to induce specific and rapid (e.g. CDK1-Tyr15), resulting in CDK activation. Thus, Cdc25 depletion of the endogenous Chk1 protein, which resulted in aberrant inhibition ends up causing cell cycle arrest (Boutros et al., 2007). – accumulation of DNA damage factors that induced cell cycle arrest at S There are three Cdc25 paralogs (Cdc25A Cdc25C) in human cells. or . Cdc25A was stabilized upon Chk1 depletion before the Cdc25A phosphorylation by Chk1 triggers its degradation in a accumulation of DNA damage factors. Simultaneous depletion of Chk1 ubiquitin/proteasome-dependent manner (Boutros et al., 2007; and Cdc25A partially suppressed the defects caused by Chk1 single Mailand et al., 2000). Phosphorylation of Cdc25B and Cdc25C by depletion. These results indicate that, similar to its function in DDR, Chk1 reduces their activity (Boutros et al., 2007). Chk1 controls normal cell cycle progression mainly by inducing DDR signaling pathways were initially considered to play critical Cdc25A degradation. roles mainly in cells under exogenous DNA-damaging stress. However, previous studies showing that complete deficiency of KEY WORDS: Chk1, Cdc25A, Auxin-inducible degron, AID, Cell cycle ATR or Chk1 leads to aberrant accumulation of DNA damage with progression early embryonic lethality (Brown and Baltimore, 2000; Liu et al., 2000; Takai et al., 2000) challenged this general belief. INTRODUCTION Interestingly, knockout of Cdc25A also results in early embryonic DNA damage caused by exogenous and endogenous factors occurs lethality (Ray et al., 2007). In sharp contrast, mice with complete constantly in normal cells. DNA lesion activates a variety of cellular deficiency of Cdc25B or Cdc25C alone or with double knockout of DNA damage response (DDR) mechanisms, including the cell cycle Cdc25B and Cdc25C are viable without any relevant phenotype, checkpoints, DNA repair, and senescence (Bartek and except for some meiotic abnormalities during oogenesis in Cdc25B- Lukas, 2007; Ciccia and Elledge, 2010; Jackson and Bartek, 2009). knockout mice (Chen et al., 2001; Ferguson et al., 2005; Lincoln The DDR requires the activation of two evolutionarily conserved et al., 2002). Cells derived from Cdc25B and Cdc25C double- protein kinase cascades, the ATM–Chk2 and ATR–Chk1 (Chk1 and knockout mice show no apparent abnormalities in their cell cycle Chk2 are encoded by CHEK1 and CHEK2, respectively, in profile or DDR (Ferguson et al., 2005). However, in human somatic mammals) pathways (Antoni et al., 2007; Awasthi et al., 2015; cells, Cdc25B reportedly plays critical roles in normal G2/M Blackford and Jackson, 2017; Flynn and Zou, 2011; Goto et al., transition and in the at the G2 phase, together 2015; Medema and Macu˚rek, 2012; Reinhardt and Yaffe, 2009; with Cdc25A (Cazales et al., 2005; Lammer et al., 1998; Timofeev Saldivar et al., 2017; Shiloh and Ziv, 2013; Zhang and Hunter, 2014). et al., 2010; Vázquez-Novelle et al., 2010). A previous study has shown that Chk1 inhibition using a Chk1

1Department of Neural Regeneration and Cell Communication, Mie University inhibitor or Chk1-specific siRNA induces accumulation of DNA Graduate School of Medicine, Tsu, Mie 514-8507, Japan. 2Department of damage and DDR even in the absence of exogenous DNA insults Science, National Institute of Genetics, Research Organization (Beck et al., 2010; Syljuasen et al., 2005). However, whether the of Information and Systems (ROIS), Mishima, Shizuoka 411-8540, Japan. 3Department of Genetics, The Graduate University for Advanced Studies signaling pathway mediated by Chk1 in unperturbed cell cycle (SOKENDAI), Mishima, Shizuoka 411-8540, Japan. 4Department of Physiology, Mie progression and in DDR is similar remains unknown. To clarify University Graduate School of Medicine, Tsu, Mie 514-8507, Japan. 5Department of this, conditional gene-knockout- or RNA interference-mediated Immunology, Mie University Graduate School of Medicine, Tsu, Mie 514-8507, Japan. knockdown of Chk1 could be used. However, it takes days to observe significant phenotypic changes using these technical *Author for correspondence ([email protected]) approaches, and thus it is extremely difficult to distinguish H.G., 0000-0002-6796-4467 whether the observed phenotype is due to Chk1 inhibition or to DDR. The use of Chk1 inhibitor is an alternative approach but, in

Received 23 July 2018; Accepted 2 January 2019 this case, specificity of the inhibitor could become a problem. To Journal of Cell Science

1 SHORT REPORT Journal of Cell Science (2019) 132, jcs223123. doi:10.1242/jcs.223123 overcome these difficulties, in the present study, we established a We examined the effect of acute Chk1 depletion on cell human colon carcinoma HCT116 cell line carrying endogenous proliferation in the absence of exogenous DNA insult. As shown Chk1 that can be rapidly and specifically degraded in an indole-3- in Fig. 2A, each Chk1–mAID cell line showed only marginal acetic acid (IAA; a natural auxin)-dependent manner (Natsume changes in after 1 day in the presence of IAA compared et al., 2016; Nishimura et al., 2009). Rapid Chk1 depletion impeded to control DMSO. However, a 3-day culture in the presence of IAA cell proliferation and caused accumulation of aberrant DNA damage significantly reduced the cell proliferation of four independent marker , such as γH2AX (H2AX phosphorylated at Ser139; Chk1–mAID cell lines but not that of their parental cells (Fig. 2A; H2AX is encoded by H2AFX). Cdc25A stabilization was observed also see Fig. 3C). FACS analyses revealed that a 3-day culture in the before the accumulation of DNA damage factors. In addition, the presence of IAA increases the sub-G1 fraction (a measure of the Chk1 depletion-associated phenotype was partially suppressed by apoptotic cell fraction) in Chk1mACl/mACl cell lines (Fig. 2B). depletion of both Chk1 and Cdc25A, suggesting that Chk1 plays a However, as judged by the Trypan Blue staining, the percentage of role in the normal cell cycle in the absence of exogenous DNA dead cells varied from less than 2% to more than 10% by experiment damage mainly by targeting Cdc25A. (data not shown; also see FACS data in Fig. 4A), suggesting that the increase in apoptosis may not be the main cause of growth defect by RESULTS AND DISCUSSION Chk1 depletion. FACS analyses also indicated that the G2/M The CRISPR/Cas9 gene editing technology enables insertion of a fraction was elevated upon IAA treatment for 3 days in DNA fragment (e.g. tags and drug-resistant markers) at a desired Chk1mACl/mACl cells but not in their parental cells (Fig. 2B). Since gene by a sequence-specific DSB induction and homologous only 1–2% mitotic cells were detected in these IAA-treated cells recombination repair (Cong et al., 2013; Mali et al., 2013; Ran et al., (Fig. S2A), Chk1 depletion induces cell cycle arrest at the G2/M 2013). We combined this gene knock-in technology to generate transition; we observed a similar tendency for the Chk1mAM/mAM human conditional cells using a donor harboring a minimum auxin- cell lines (data not shown). A 3-day culture in the presence of IAA inducible degron (mAID) tag (Natsume and Kanemaki, 2017; increased the level of p53 and p21 proteins but not the level of Natsume et al., 2016; Nishimura et al., 2009). As shown in Fig. 1A–C, CDK1 phosphorylated at Tyr15 (an inactive form of CDK1; we established a human near-diploid HCT116 colon carcinoma cell Fig. S2B) in Chk1–mAID cell lines. Therefore, Chk1 depletion line (Thompson and Compton, 2008) expressing an auxin-inducible results in p53 stabilization, which induces cell cycle arrest likely ubiquitin ligase component, Oryza sativa (Os)TIR1, and the through the induction of CDK inhibitor(s), such as p21. endogenous Chk1 protein fused with mAID and a monomeric We analyzed early changes after IAA treatment by Clover (mAID–mClover; mACl) (Natsume et al., 2016) or with immunoblotting (Fig. 2C; Fig. S3). The phosphorylation of mAID fused to five repeats of the Myc epitope tag (mAID–5Myc; H2AX-Ser139 (γH2AX) and Chk2-Thr68 gradually increased mAM) at its C-terminus. We established two independent clones per after IAA incubation; it peaked at ∼20–24 h in the observed range each genotype. Even in the absence of IAA, the protein level of (from 0 h to 24 h). Since p53 stabilization was detected just after this mAID-tagged Chk1 was slightly lower than that of the wild-type elevation, DNA damage accumulated upon the rapid Chk1 (WT) Chk1 (Fig. 1C, see the lanes corresponding to DMSO-treated depletion, which in turn induced p53 stabilization. By contrast, cells, labeled D). This was likely due to weak activation of OsTIR1 the protein level of Cdc25A started to increase at ∼2–4 h, peaked at in the absence of IAA (Natsume et al., 2016). However, cells ∼8 h and then decreased thereafter (Fig. 2C; Fig. S3; also see expressing mAID-tagged Chk1 (Chk1–mAID cells) showed only a Fig. 1D). These observations suggest that acute Chk1 depletion marginal growth defect in the absence of IAA (data not shown; also leads to Cdc25A stabilization before accumulation of DNA damage see Fig. 2A), suggesting that the Chk1 fusions are functional and and expression of DDR proteins. that the expression level is sufficient to support proliferation. Since DNA damage products such as γH2AX reportedly start to The addition of IAA induced depletion of Chk1, but not of Chk2 increase 1 h after incubation with the Chk1 inhibitor UCN-01 in in the established Chk1–mAID cell lines [Fig. 1C, compare IAA- U2OS cells (Syljuasen et al., 2005), we used two types of Chk1 treated cells (labeled I) with DMSO-treated cells (labeled D)]. The inhibitors, MK-8776 (Thompson et al., 2012; Chang and Eastman, mAID-tagged Chk1 was rapidly depleted within 15–30 min after 2012) and CHIR-124 (Tao et al., 2009; Tse et al., 2007). In HCT116 the addition of IAA to the growing medium, as shown for clone 1 cells, incubation with 2 µM MK-8776 stabilized Cdc25A, similar to and clone 3 in Fig. 1D. We confirmed similar depletion kinetics in what was observed with IAA-induced Chk1 depletion (compare clone 2 and clone 4 (data not shown; also see Fig. 2C). Fig. S2C with Figs 1D, 2C and Fig. S3); however, we observed no In response to replication stress induced by hydroxyurea (HU) significant elevation of γH2AX (data not shown). In HCT116 and treatment or to DNA damage from UV irradiation, mAID-tagged HeLa cells, Cdc25A stabilized at ∼2–4 h after the treatment Chk1 was phosphorylated at Ser317 and Ser345 by ATR (Zhao and with 250 nM CHIR-124 whereas it took ∼8 h to detect γH2AX Piwnica-Worms, 2001) and at Ser296 through Chk1 auto- (Fig. S2D); these kinetics closely resemble data obtained using phosphorylation (Clarke and Clarke, 2005; Kasahara et al., 2010), Chk1–mAID cell lines (Fig. 2C; Fig. S3). We could be observing a in similar manner to that seen for the WT Chk1 protein (Fig. S1A–C). time lag (∼2–4 h) between Chk1 inhibition and Cdc25A In the absence of IAA, UV irradiation reduced the Cdc25A protein stabilization because the Cdc25A degradation triggered by Chk1- level in Chk1–mAID cell lines, as in their parental cells (Chk1WT/WT; induced phosphorylation requires multiple steps, including Fig. S1D). On the other hand, IAA incubation abolished this Cdc25A additional Cdc25A phosphorylation and ubiquitylation (Boutros degradation in Chk1–mAID cell lines, but not in their parental cells et al., 2007; Goto et al., 2015). On the other hand, the kinetics of (Fig. S1D), consistent with previous reports showing that Chk1 γH2AX appears to differ slightly among experimental settings, phosphorylates Cdc25A and that this mediates Cdc25A degradation which may reflect a difference in the degree of Chk1 inhibition and/ during the DDR (Mailand et al., 2000; Zhao et al., 2002). This Chk1 or off-target effect(s). phosphorylation (Fig. S1A–C) and Cdc25A degradation (Fig. S1D) In order to further clarify the role of Cdc25A in cell proliferation, indicate that the functions of mAID-tagged Chk1 and WT Chk1 we tagged endogenous Cdc25A with mAID and mCherry2 (mACh) WT/WT mACl/mACl protein in the DDR are similar. at the C-terminus in the Chk1 or Chk1 backgrounds Journal of Cell Science

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Fig. 1. Establishment of a rapid AID system for depletion of endogenous Chk1. (A) Strategy for the insertion of mAID–mClover (mACl) or -5Myc (mAM) coding sequences just upstream of the termination codon on exon 12 of the human Chk1 loci in HCT116 cells stably expressing OsTIR1 (HCT116-OsTIR1s). Each targeting vector contains a mACl or mAM tag (indicated as black boxes) plus a Neo- or Hyg-resistant gene cassette (black boxes with gradation) between 5′ and 3′ homology arms (bold lines) flanking the DSB site (scissors). The expected genomic configuration after homologous recombination repair is shown on the right. Each primer position for genomic PCR (gPCR) is also indicated in the cartoon. (B,C) Established clones were screened by gPCR (B) and immunoblotting (C). In C, each clone was cultured in the presence of DMSO (labeled D) or 500 μM IAA (labeled I) for 4 h. B and C indicate successful modification with the coding sequence and rapid degradation of mAID- tagged Chk1 after IAA addition, respectively. (D) Time course showing the presence of the mAID-tagged Chk1 after IAA addition. This finding indicates Cdc25A stabilization after Chk1 degradation.

(Fig. S4A; Fig. 3A). Before generating the double mAID cell lines Cdc25A-depleted cells. The low proliferation rate seen upon Chk1 (Chk1mACl/mACl+Cdc25AmACh/mACh), we removed the floxed depletion was partly improved by co-depletion of Cdc25A. Hyg-resistant cassette from Chk1mACl/mACl cells (clone 3) with Cre These observations also raised the question of whether prior recombinase (see Fig. 1A). From this intermediate clone (referred to Cdc25A degradation completely rescues growth defect caused by as Chk1mACl/mACl clone 3′), we generated the double mAID cell lines Chk1 inhibition. To address this question, Cdc25AmACh/mACh cells (Chk1mACl/mACl+Cdc25AmACh/mACh clones 5 and 6; Fig. 3A). (clone 8) were pre-treated with DMSO or IAA for 2 h and then Simultaneous depletion of Chk1 and Cdc25A partially suppressed incubated with 0–750 nM CHIR-124 for a further 3 days (Fig. S4C). the cell growth defect observed in Chk1-depleted cells (Fig. 3B,C). Prior degradation of Cdc25A did not completely recover the growth Cdc25A single depletion was not associated with a growth advantage inhibition caused by CHIR-124 treatment (Fig. S4D). However, the (Fig. S4B; Fig. 3C) because the growth defect was prominent in both growth defect caused by Cdc25A depletion was partly attenuated by Cdc25AmACh/mACh cell lines at late stages (at ∼6 days after the co-treatment with 250–500 nM CHIR-124 (Fig. S4D,E); we addition of IAA; data not shown). Therefore, Cdc25A counteracts observed a similar tendency using clone 7 (data not shown). Chk1 even in normal cell cycle progression. Our current observations These findings support the idea that there is a close relationship closely resemble the results reported in a previous study that used between Cdc25A and Chk1. Chk1- and/or Cdc25A-specific siRNA(s) in U2OS or TIG-3-tert cells Finally, we performed the immunoblotting (Fig. 3D) or FACS (Beck et al., 2010). Cell growth was impaired by single depletion analyses after pulse BrdU labeling (Fig. 4). A 3-day incubation in of Chk1 or Cdc25A but the phenotype was far milder in the presence of IAA elevated the levels of p53 and p21 not only in Journal of Cell Science

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Fig. 2. Endogenous Chk1 degradation induces Cdc25A stabilization prior to accumulation of DNA damage. (A) Each clone was incubated in a growing medium containing DMSO or IAA for the indicated number of days. The growth curve of each clone was obtained as described in the Materials and Methods. Data represents mean± s.e.m. from three independent experiments. These results indicate growth defects after Chk1 degradation. (B) Each clone was incubated in the presence of DMSO or IAA for 3 days. The vertical and horizontal axis indicates cell counts and propidium iodide (PI) intensity, respectively. The position of one copy (1C) or two copies (2C) of genetic information is also indicated. The FACS data indicate the increase in sub-G1 and G2/M fractions. (C) Time-dependent changes of the indicated markers after adding IAA to the growing medium. DNA damage protein markers were elevated after Cdc25A stabilization.

Chk1-mACl cells (clone 3′) but also in double mAID cells (clones 5 Even in the absence of any exogenous DNA insult, Chk1 is and 6; Fig. 3D). Both the mitotic index (Fig. S2A) and the FACS reportedly required to control DNA replication: Chk1 restricts the data (Fig. 4A,B) revealed arrest of the above cells (clones 3′, 5 and number of firing replication origins (Beck et al., 2010; Maya- 6) at G2 phase after the 3-day incubation in the presence of IAA. Mendoza et al., 2007; Saldivar et al., 2018; Syljuasen et al., 2005; The Chk1-depleted cells also arrested at because the Zhang and Hunter, 2014). Treatment with Chk1 inhibitor, Chk1- proportion of BrdU-negative S phase cells was elevated among the specific siRNA or disruption of the Chk1-gene activates latent DNA total S phase cells (Fig. 4A,C); however, the population of cells replication origins (e.g. late origin firing) leading to depletion of arrested at G2 phase was much higher (Fig. 4). Since co-depletion of replication factors at each replicon. This results in stalled DNA Cdc25A partially attenuated all these parameters linked to the cell replication forks that induce DNA breaks. In this study, we cycle arrest caused by Chk1 depletion (Figs 3D and 4), we can infer established HCT116 cell lines carrying endogenous Chk1 tagged that Chk1 controls normal cell cycle progression mainly by with a mAID tag at its C-terminus. Using these cell lines, we found inducing Cdc25A degradation. that Chk1 depletion induces cell cycle arrest at the S or G2 phase but Journal of Cell Science

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Fig. 3. Co-induction of Cdc25A degradation partially attenuates the growth defect induced by Chk1 degradation. (A) Establishment of double mAID (Chk1mACl/mACl+ Cdc25AmACh/mACh) cell lines. An asterisk indicates the position of degraded Cdc25A–mACh. (B) Growth curve of each clone in the growing medium containing DMSO (labeled D) or IAA (labeled I). (C) Each cell line was incubated in the presence of DMSO or IAA for 3 days. The bar graph indicates the ratio of the number of IAA-treated cells to DMSO-treated cells. Data represents mean±s.e.m. from nine (parent cells) or six (other cell lines) independent experiments. *P<0.05, **P<0.01, ***P<0.001 (black asterisks, two- tailed, unpaired t-test compared to parent cells; red asterisks, two-tailed, unpaired t-test compared to clone 3′). (D) Each clone was cultured in the presence of DMSO (labeled D) or IAA (labeled I) for 3 days and immunoblotted for the indicated proteins. At the bottom, the relative band intensity of the anti-p53 signal is indicated as fold increase in relation to the band intensity in Chk1mACl/mACl cells (clone 3′) treated with DMSO (a bar graph). Data represent the mean±s.d. of three independent experiments.

not at the or G1/S transition. These results are consistent siRNA(s) (Beck et al., 2010). These observations suggest that Chk1 with the observations showing that Chk1 inhibition mainly disturbs controls normal cellular proliferation through a signaling pathway DNA replication during a normal cell cycle progression leading to similar to the DDR. The ability of ChK1 to phosphorylate several accumulation of DNA damage specifically at S or G2 phase (Beck substrates other than Cdc25A during the DDR (Dai and Grant, 2010; et al., 2010; Maya-Mendoza et al., 2007; Saldivar et al., 2018; Patil et al., 2013; Zhang and Hunter, 2014) might explain why co- Syljuasen et al., 2005). In addition, we demonstrated that rapid and depletion of Chk1 and Cdc25A did not completely suppress the specific Chk1 depletion induces Cdc25A stabilization before DNA growth defect. These issues will be addressed in future investigations. damage accumulation. This finding is in line with previous reports showing Cdc25A stabilization after treatment with a Chk1 inhibitor MATERIALS AND METHODS (Sørensen et al., 2003) and in Chk1-haploinsuffient mice (Lam et al., Genomic PCR 2004). In addition, co-depletion of Chk1 and Cdc25A partially Genomic DNA (gDNA) was purified from HCT116 cells stably expressing suppressed the growth defect observed after Chk1 single depletion, Oryza sativa (Os)TIR1 (HCT116-OsTIR1s) (Natsume et al., 2016) and their confirming a previous report using Chk1- and/or Cdc25A-specific derivatives by using a Wizard® genomic DNA purification kit (Promega, Journal of Cell Science

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Fig. 4. Co-induction of Cdc25A degradation partially rescues the cell cycle arrest induced by Chk1 degradation. FACS analyses after pulse BrdU labeling. (A) The vertical axis indicates the anti-BrdU intensity (left) or cell counts (right) as shown in the example at the top. (B–D) Graphs of the percentage of cells in G2/M, the percentage of BrdU-negative S phase cells of the total S phase cells, and the percentage of cells that are either BrdU-negative S phase or in G2/M. Similar results were observed in two other independent experiments (data not shown).

Madison, WI). Genomic PCR (gPCR) was amplified using Tks Gflex DNA Gene targeting polymerase (Takara Bio, Kusatsu, Shiga, Japan) (Natsume et al., 2016) with Gene targeting using CRISPR/Cas9 was performed in HCT116-OsTIR1s the following primers: 5′-AGCATTTGCCGCAGTACTCT-3′ (Chk1 sense and their derivatives as described previously (Natsume et al., 2016). In brief, primer, denoted S) and 5′-GCTTCGCTTCACAGACTGA-3′ (Chk1 annealed DNA oligonucleotides for each guide RNA (gRNA) was cloned antisense primer, denoted AS) for the Chk1 gene; 5′-GTTTCAGG- into BbsI sites in pSpCas9 (BB)-2A-Puro V2.0 (pX459, Addgene plasmid TTCAGGGGGAGG-3′ (3′UTR antisense primer, denoted 3′ UTR), 5′- ID #62988) (Ran et al., 2013). The gRNA sequences targeting Chk1 and TCGATCAGGATGATCTGGAC-3′ (Neo sense primer, denoted Neo) and Cdc25A loci were 5′-GAGCCGATGGTCCGATCATG(TGG)-3′ and 5′- 5′-CATATGCGCGATTGCTGATC-3′ (Hyg sense primer, denoted Hyg GAAGAAGCTCTGAGGGCGGC(AGG)-3′, respectively [protospacer in Fig. 1A) for the inserted DNA cassette described below; and 5′- adjacent motif (PAM) sequences are indicated in parentheses]. For the GGTGCCGGTATGTGAGAGAG-3′ (Cdc25A sense primer) and 5′- construction of the Chk1-targeting vectors, 5′- and 3′-homology arms were GCAACATTCCAGCACTGAGC-3′ (Cdc25A antisense primer) for the amplified from the Chk1 gPCR product. By using a NEBuilder HiFi DNA

Cdc25A gene. assembly master mix (New England Biolabs, Ipswich, MA), these arms Journal of Cell Science

6 SHORT REPORT Journal of Cell Science (2019) 132, jcs223123. doi:10.1242/jcs.223123 were inserted into the pMK289 [mAID-mClover (mACl)-Neo resistance anti-p21, -γH2AX or -GAPDH antibodies, we used immunoreaction cassette], pMK290 (mACl-Hyg resistance cassette), and their modified enhancer solution (Can Get Signal®, Toyobo, Osaka, Japan) for the vectors [mAID-5Myc (mAM)-Neo and -Hyg resistance cassettes; also dilution of primary and secondary antibodies. The band intensity in the see Fig. 1A] as described previously (Natsume et al., 2016). For the immunoblotting was quantified using densitometry as described previously generation of Chk1mACl (mAM)/mACl (mAM) cells, HCT116-OsTIR1 s were (Li et al., 2012). co-transfected with Chk1-pX459 plasmid and (modified) pMK289- and pMK290-Chk1-targeting vectors (1:1:1 ratio). After selection with Acknowledgements 0.4 mg/ml G418 and 0.1 mg/ml Hygromycin B Gold (Nacalai Tesque, We would like to thank Y. Hayashi, N. Tanigawa and Y. Nakai (Aichi Center Kyoto, Japan), each clone was screened by gPCR and immunoblotting. Research Institute) for technical assistance; M. Fujita (Kyushu University), K. Furuya For the Cdc25A gene, we employed similar strategies, except for the use (Kyoto University) and H. Kosako (Tokushima University) for helpful discussion. of pMK292 [mAID–mCherry2 (mACh)-Neo resistance cassette]- and pMK293 (mACh-Hyg resistance cassette)-Cdc25A-targeting vectors Competing interests The authors declare no competing or financial interests. (Natsume et al., 2016). For the generation of double mAID (Chk1mACl/mACl+Cdc25AmACh/mACh) cell lines, the floxed Hyg resistance mACl/mACl Author contributions cassette (also see Fig. 1A) was removed from Chk1 cells (clone 3) Conceptualization: H.G.; Methodology: H.G., T.N., M.T.K.; Validation: H.G.; through infection with adenovirus carrying Cre recombinase. The Formal analysis: H.G., T.N., M.T.K.; Investigation: H.G., T.N., M.T.K., A.K., S.W.; established cells (clone 3′) were then transfected with Cdc25A-pX459 Resources: H.G., T.N., M.T.K., A.K., S.W.; Data curation: H.G., T.N., M.T.K.; plasmid and pMK293-Cdc25A-targeting vectors (1:2 ratio). Writing - original draft: H.G.; Writing - review & editing: H.G., M.T.K., E.C.G., A.M.; Visualization: H.G.; Supervision: H.G.; Funding acquisition: H.G., M.I., A.M. Cell culture Original HCT116 or HeLa cells were purchased from the American Type Funding This work was supported in part by Grants-in-Aid from the Ministry of Education, Culture Collection (ATCC CCL-247 or CCL-2, respectively). HCT116 cell Culture, Sports, Science, and Technology of Japan [the Japan Society for the lines including HCT116-OsTIR1 and their derivatives were cultured as Promotion of Science KAKENHI grant nos. 15K08324 and 18K06927 (H.G.); previously described (Natsume et al., 2016). HeLa cells were grown in 17K15068 (T.N.); 16K15095, 18H02170 and 18H04719 (M.T.K.); 15K09170 Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum. (E.C.G.); 15H02398 (M.I.); and 16H06461 (A.M.)]; by the Takeda Science For the induction of degradation of each mAID-tagged endogenous protein, Foundation (H.G., M.I. and A.M.); and by the Naito Foundation (M.I.). a final concentration of 500 μM IAA was added to the growing medium. An equal volume of DMSO was used as a negative control. Supplementary information All cell lines used in this study tested negative for mycoplasma Supplementary information available online at contamination. http://jcs.biologists.org/lookup/doi/10.1242/jcs.223123.supplemental References Cell growth curve Antoni, L., Sodha, N., Collins, I. and Garrett, M. D. (2007). CHK2 kinase: cancer One day before analysis of the cell proliferation rate (day −1), susceptibility and cancer therapy - two sides of the same coin? Nat. Rev. Cancer logarithmically growing cells were trypsinized and re-plated at a density 7, 925-936. of 5×104 cells per dish. One day after re-plating (day 0), cells were incubated Awasthi, P., Foiani, M. and Kumar, A. (2015). ATM and ATR signaling at a glance. in a culture medium containing DMSO or 500 μM IAA for the number of J. Cell Sci. 128, 4255-4262. Bartek, J. and Lukas, J. (2007). DNA damage checkpoints: from initiation to indicated days. Each medium was changed every 2 days. The number of recovery or adaptation. Curr. Opin. Cell Biol. 19, 238-245. living cells was quantified using a TC20 automated cell counter (Bio-Rad Beck, H., Nähse, V., Larsen, M. S. Y., Groth, P., Clancy, T., Lees, M., Jørgensen, Laboratories, Richmond, CA). In brief, cells were trypsinized and M., Helleday, T., Syljuåsen, R. G. and Sørensen, C. S. (2010). Regulators of resuspended in the culture medium at each time point. Then, 10 µl of cell cyclin-dependent kinases are crucial for maintaining genome integrity in S phase. suspension was mixed with an equal volume of 0.4% Trypan Blue solution J. Cell Biol. 188, 629-638. (Bio-Rad Laboratories). The mixture was applied to the cell counter Blackford, A. N. and Jackson, S. P. (2017). ATM, ATR, and DNA-PK: the trinity at ’ the heart of the DNA damage response. Mol. Cell 66, 801-817. following the manufacturer s protocol. Boutros, R., Lobjois, V. and Ducommun, B. (2007). CDC25 phosphatases in cancer cells: key players? Good targets? Nat. Rev. Cancer 7, 495-507. Flow cytometry Brown, E. J. and Baltimore, D. (2000). ATR disruption leads to chromosomal FACS analyses were performed as described previously (Natsume et al., fragmentation and early embryonic lethality. Dev. 14, 397-402. 2017), except that cells were fixed in 95% ethanol. Cazales, M., Schmitt, E., Montembault, E., Dozier, C., Prigent, C. and Ducommun, B. (2005). CDC25B phosphorylation by Aurora A occurs at the G2/M transition and is inhibited by DNA damage. Cell Cycle 4, 1233-1238. Immunoblotting Chang, L.-J. and Eastman, A. (2012). Decreased translation of p21waf1 mRNA Immunoblotting analyses were performed as previously described causes attenuated p53 signaling in some p53 wild-type tumors. Cell Cycle 11, (Enomoto et al., 2009; Goto et al., 2016). We used the following 1818-1826. antibodies: Chk1, 1:2000 [mouse monoclonal antibody (mAb), 2G1D5; Chen, M.-S., Hurov, J., White, L. S., Woodford-Thomas, T. and Piwnica-Worms, cat. no. #2360, Cell Signaling Technology (CST), Beverly, MA]; Chk1- H. (2001). Absence of apparent phenotype in mice lacking Cdc25C protein pS296, 1:2000 (rabbit mAb, D309F; cat. no. #90178, CST); Chk1-pS317, phosphatase. Mol. Cell. Biol. 21, 3853-3861. ® Ciccia, A. and Elledge, S. J. (2010). The DNA damage response: making it safe to 1:2000 (XP rabbit mAb, D12H3; cat. no. #12302, CST); Chk1-pS345, play with knives. Mol. Cell 40, 179-204. ® 1:2000 (rabbit mAb, 133D3; cat. no. #2348, CST); Chk2, 1:2000 (XP Clarke, C. A. L. and Clarke, P. R. (2005). DNA-dependent phosphorylation of Chk1 rabbit mAb, D9C6; cat. no. #6334, CST); Chk2-pT68, 1:2000 (rabbit mAb, and Claspin in a human cell-free system. Biochem. J. 388, 705-712. C13C1; cat. no. #2197, CST); p21 Waf1/Cip1, 1:2000 (rabbit mAb, 12D1; Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D., Wu, X., cat. no. #2947, CST); γH2AX, 1:2000 (rabbit mAb, 20E3; cat. no. #9718, Jiang, W., Marraffini, L. A. et al. (2013). Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819-823. CST); CDK1-pY15, 1:2000 (rabbit mAb, 10A11; cat. no. #4359, CST); Dai, Y. and Grant, S. (2010). New insights into checkpoint kinase 1 in the DNA Cdc25A, 1:500 (mouse mAb, F6; cat. no. sc-7389, Santa Cruz damage response signaling network. Clin. Cancer Res. 16, 376-383. Biotechnology, Santa Cruz, CA); p53, 1:2000 (mouse mAb, 80/p53; cat. Enomoto, M., Goto, H., Tomono, Y., Kasahara, K., Tsujimura, K., Kiyono, T. and no. 610184, BD Transduction Laboratories, San Diego, CA); GAPDH, Inagaki, M. (2009). Novel positive feedback loop between Cdk1 and Chk1 in the 1:10,000 (mouse mAb, 5A12; cat. no. 010-25521, Wako Pure Chemical nucleus during G2/M transition. J. Biol. Chem. 284, 34223-34230. Industries, Osaka, Japan); horseradish peroxidase (HRP)-conjugated anti- Ferguson, A. M., White, L. S., Donovan, P. J. and Piwnica-Worms, H. (2005). Normal cell cycle and checkpoint responses in mice and cells lacking Cdc25B and rabbit-IgG, 1:50,000 [goat polyclonal antibody (pAb); cat. no. ab205718, Cdc25C protein phosphatases. Mol. Cell. Biol. 25, 2853-2860. Abcam, Cambridge, MA]; and HRP-conjugated anti-mouse-IgG, 1:50,000 Flynn, R. L. and Zou, L. (2011). ATR: a master conductor of cellular responses to

(rabbit pAb; cat. no. ab97046, Abcam). Except for the immunoblotting with DNA replication stress. Trends Biochem. Sci. 36, 133-140. Journal of Cell Science

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